Fig. 6

Left biased lateralization of FGF pathway activation depends on lateralized Nodal signaling. (A–F) Confocal sections of the pineal complex at 30 hpf showing the expression of Tg(dusp6:d2EGFP) (green) in two illustrative control embryos (A and B; n = 36) and in two embryos injected with no tail morpholinos (ntl MO) (C and D; n = 37) or southpaw morpholinos (spaw MO) (E and F; n = 38); the superimposed nuclear staining (gray) allows visualization of the epiphysis (white circle) and parapineal (yellow circle). Embryo view is dorsal, anterior is up. (Scale bars, 10 µm.) (G–I′) Polar graphs showing the distribution and mean number of total (gray) or Tg(dusp6:d2EGFP)⁺ (green) parapineal cells in each 45 °C hemiquadrant relative to the parapineal mean position (graph center) at 30 hpf in control embryos (G–G″, n = 36), ntl morphants (H–H″; n = 37), or spaw morphants (I–I″; n = 38). For each context, cell distributions and mean numbers of cells are averaged within embryos that display an AI (see Materials and Methods and SI Appendix) in Tg(dusp6:d2EGFP) expression ≤ −0.2 (G, n = 28 of 36; H, n = 14 of 37; I, n = 13/38), AI ≥ +0.2 (G″, n = 4 of 36; H″, n = 11 of 37; I′, n = 13 of 38) or −0.2 ≤ AI ≥ +0.2 (G′, n = 4 of 36; H′, n = 12 of 37; G″, n = 12 of 38); percentages of embryos in each category are shown on the lower right side of the polar graph. Radial axis (scale 0–6) represents the mean number of Tg(dusp6:d2EGFP)⁺ parapineal cells (green area) or all parapineal cells (gray area) per hemiquadrant and per embryo. In both ntl and spaw morphants, the expression of Tg(dusp6:d2EGFP) is either not clearly lateralized or lateralized with random orientation (enriched on the left or on the right side). (J) Dot plot showing, for each embryo, the AI in the number of Tg(dusp6:d2EGFP)⁺ cells in the left posterior versus the right posterior quadrant in control embryos (n = 36), ntl morphants (n = 37) or spaw morphants (n = 38). Left-sided lateralization of Tg(dusp6:d2EGFP) expression observed in controls is lost in both ntl and spaw morphants. The distribution of AIs is less spread in spaw morphants than in ntl morphants (P = 0.045 in F test comparing variances) showing that Tg(dusp6:d2EGFP) expression is overall less lateralized in spaw morphants than in ntl morphants. Gray-shaded zone shows AI between −0.2 and +0.2. (K) Dot plot showing, for each embryo, the number of Tg(dusp6:d2EGFP)⁺ parapineal cells in control embryos, ntl morphants and spaw morphants at 30 hpf. The number of Tg(dusp6:d2EGFP)⁺ cells is overall increased in the parapineal of spaw morphants compared with controls (P = 0.026, Welsh t test). In J and K, means ± SD are indicated as a horizontal (mean) and vertical lines (SD); statistical significance is indicated, *P < 0.05. (L) Dot plot showing, for each embryo, the mean parapineal position in micrometers distant to the brain midline (x = 0) in control embryos, ntl morphants and spaw morphants at 30 hpf. Gray-shaded zone shows parapineal mean position between −5 µm and +5 µm. In controls and ntl morphants, the parapineal had usually started to migrate, respectively, toward the left or randomly by 30 hpf, while it had not initiated migration in spaw morphants at this stage.